Amicetin a and its salts



Oct. 20, 1959 Filed Aug. 13, 1953 FIGURE 2 C. DE BOER El" AL AMICETIN A AND ITS SALTS HONVLLIWSNVUL WAVE NUMBERS IN cm" H l2 WAVE LENGTH IN MICRONS INFRARED ABSORPTION SPECTRUM I500 I400 I300 WAVE NuMaERs m CM" 2500 WAVE LENGTH IN MICRONS 2 Sheets-Sheet 2 AMIGETIN A CLARENCE DE BOER JACK W. HINMAN INVENTORS United States Patent AMICETIN A AND ITS SALTS Patented Oct. 20, 1959 Standards in Nomenclature. All seeding was done with a spore suspension, the test tubes containing the various culture media being incubated between 24 and 28 degrees centigrade. Readings were taken on the 4th, 7th, 15th and 22nd days.

Clarence De Boer and Jack W. Hinman, Kalamazoo PP Produces 31mg, filamentous y- Township, Kalamazoo County, Mich., assignors to The celium, which branches profusely, and conidia in sinis- Upjohn Company, Kalamazoo, Mich., a corporation trorse spiral chains. The spirals occur singly or in clusters of Michigan and measure fifteen to twenty microns in length by three 10 to five microns in width Most of the spirals consist of nal No. 374 111 Apphcatlon August 1953 Se three to four volutions. The hyphae are approximately 5 Claims. (Cl. 167-65) 1.0 micron in width while the conidia are between 1.5 and p 2.0 microns in diameter.

Although S. vinaceus-drappus is similar in some re- This invention relates to a new and useful antibiotic 15 spects to Streptomyces lavendulae, these microorganisms substance, amicetin A, obtained by the cultivation of are y dlstlngulshable not y by their differences Streptomyces vinaceus-drappus, a microorganism undein color (lavender 'vs. vinaceous-drab) but also by disscribed hitherto. tlnct differences in their cultural characteristics. These This application is a continuation-in-part of copending 9 dliferences are Set forth In the following table! application, Serial No. 238,072, filed July 23, 19 5l.

It is an object of the present invention to provide a new 5'. Zavendulae S. rinaceus-drappus and useful antibiotic substance possessing bacteriostat c or bacteriocidal activity against certain acid-fast bacteria 3 :1 water-soluble mycelialpigno mycellal pigment.

an m Partlcularly, 3(i l j lf l 9 l gf 05 0210;) change of gelatin from cream no color change of gelatin.

this invention is to mow e a su s ance w c s a ve 0 rown.

Little owth on otato slantheav owth on otato slant aga s gramnegatwe and P bactena' other little liange in col ar. vin iac t ius-drab in olor. objects and features of the invention Wlll be apparent to Oval ped comdia spherical shaped conidia, those skilled in the art to which this invention pertains.

TABLE I Cultural characteristics of Streptomyces vinaceus-drappus Medium Amount of Color of Aerial Mycelium and Soluble Remarks Growth Spores Pigment Casein Starch agar Profuse--- Light vlnaceous drab None ctli loiriesl filat, smooth, entire. Partial starch y o ys s.

Czapeks-Dox agar Moderate- Pallidv naceous drab do Cinnamon bufi reverse.

Irish potato Heavy-- P l 71 11 rab- -.do. Rugoile11 iliirface. Exceptional growth and SP0 8. on.

s t tato rio Light vinaceous drab" do Wartymsnirface. Excellent growth and good spor 3. on.

Nutrient agar Light..-- 'W11lt do.-.-- Chamois reverse.

Dextrose agar Heavy-- Quaker drabdo.- Blackish-brown(2)'reverse.

Tyrosine agar" Trace"--- No sporulation do. Negative tyrosinase reaction.

Calcium malate Moderate. Pale vmaceons drab d Cellulose broth Light-.-" Pallld vinaceous drab- .do.- No decomposition of cellulose.

Starch agar-.- Moderate- W e do.-. Partial hydrolysis. Honey yellow reverse.

Nutrient brot Slight.-... White-pallid vinaceous drab.. do. Vegetative submerged growth.

Dextrose broth. Moderate- Whi e do Do.

. Nitrate brothdo -d0 do Reduces nitrates to nltrities.

Litmus milk Light--. Pallid vmaceous drab do.- Alkaline reaction, ring-like growth at surface.

Gradual reduction, no visible coagulation. Gelatin.-." Ivory.. Slow liquefaction, pellicle growth.

The antibiotic of this invention has not yet been proved to have value in human therapy.

It has been found that by cultivating, under controlled conditions, a hitherto undescribed species of microorganism referred to as Streptomyces vinaceus-drappus, a

novel antibiotic, amicetin A, is obtained. This micro organism was isolated from a sample of soil taken in Kalamazoo, Michigan. The chosen name of the new microorganism, Streptomyces vinaceus-drappus, characterizes the drab wine .color (according to Ridgway, Color Standards in Nomenclature) of its aerial mycelium and spores when cultured on a variety of media as set forth below. A culture of the living organism has been de posited with the Fermentation Division of the Northern Regional Research Laboratory at Peoria, Illinois, and has been added to its permanent collection as NRRL 2363 A careful study of the morphology and physiology of S. vinaceus-drappus showed it to be distinctly different from any previously described species of Streptomyces. The description of the microorganism, according to Bergeys Manual of Determinative Bacteriology, 6th edition, pages 929 to 933, is given below in tabular form,

the reported colors being based onkidgways Color The invention is not to be limited to the production of amicetin A by S. vinaceus-drappus or to organisms fully answering the above description which are given merely for illustrative purposes only. Other microorganisms, i.e., mutants produced from S. vinaceus-drappus by exposure to various mutating agents such as X-radiation, ultraviolet light, high-speed electrons, nitrogen mustards, and the like, can also produce amicetin A.

Amicetin A can be obtained in either the amorphous or crystalline form. Amorphous amicetin A can be converted to the crystalline material by extracting an aqueous solution thereof with a solvent such as butanol, or the like, at a pH ranging between about 7.5 and about 9.0, separating the organic solvent phase, and extracting vtribution technique of Craig, J. Biol. Chem., 155, 519 (1944).

Crystallization of amicetin A from aqueous solutions thereof -'or solutions containing some water generally yields a hydrated crystalline material. The amicetin A can be obtained in anhydrous form by drying the hyldrated material at slightly elevated temperatures in vacuo or by crystallizing the material from a substantially anhydrous solvent such as obsolute methanol, ethanol, or the like.

Amicetin A usually separates from aqueous or predominantly aqueous solutions thereof in the form of tiny needles or clusters of needles. When collected on a filter, these crystals pack tightly together to form a glossy mat. After drying in a vacuum desiccator over anhydrous magnesium perchlorate at about room temperature, the hydrated crystals of amicetin A thusobtained melt between 156 and 159 degrees centigrade, depending upon the size of crystals and the rate of heating.

Anhydrous crystals of amicetin A are obtained, as indicated above, by recrystallization of the' hydrated crystalline material from substantially anhydrous solvents such as absolute methanol, ethanol, or the like, or by drying the hydrated product in vacuo to remove the water of crystallization. On recrystallizing the antibiotic from a substantially anhydrous methanol solution, the small, dense, anhydrous plates thus-obtained melt between 209 and 212 degrees centigrade.

Amicetin A is eifective not only against various mycobacteria, notably M. tuberculosis (H37Rv), but italso inhibits the growth of gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, and the like. The spectrum of amicetin A as compared with other clinically important antibiotics which show anti-mycobacterial activity is shown below:

solutions.

Acute toxicity of antibiotics [Mg. per kg. in mice] Amicetin A is a weakly basic compound. The crystalline free base is soluble in distilled water to the extent of only about 0.1 percent but is readily soluble in acid The crystalline hydrochlorideof amicetin A may be obtained by dissolving the free base 'in a dilute hydrochloric acid solution, concentrating the solution to a small volume under reduced pressure and precipitating the resulting crystalline amicetin A hydrochloride by careful addition of acetone or a mixture of methanol and acetone. Alternatively, the aqueous solution of the hydrochloride may be freeze-dried and the resulting amorphous solid crystallized from methanol and acetone.

The crystalline amicetin A hydrochloride is thus obtained The ultraviolet absorption spectrum of anhydrous crystalline amicetin A is shown in Fig. 1. When the spec- TABLE II Microgrums of antibiotic per milliliter of nutrient broth necessary to inhibit growth of microorganisms Bacteria amicetin 1 AmicetlnA Strepto- Strepto- Neomyctn Terra- Aurep- Chlorammycin thriciu rnycin mycin phenicol A. Acid-fast: V

Mycobacterium tuberculosis v. hominis (607)-.. 1. 0 5.0 v 1.0 2. 0 0. 4 1.0 0.2 3, 3 Mycoboctcrium tuberculosis v. llominis H37Rv. 0. 2-0. 5 5-10 6. 25 Y 12. 5-50 6. 25-12. 5 25 '25-100 6. 25-12. 5 Mycobocterium avium (7992) 1.0 2.0 1.0 2.0 0:4 0.2 0 1 2.0 B. Gram-positive: V V

Staphylococcus aureus (FDA-209).- 4. 0 20 0.1 0. 2 0.02 1.10 13 3. 3 Bacillus subtilis (111.) 4. 0 20 0. 4 0. 4 r 0. 01. 10 33 1, 7 C. Gram-negative:

Escherichia coli (26) 20 1.0 1.0 0.1 2 5 1.0 1. 7 Klcbstella pueumom'ae (10031) 20 20 0. 1 0.2 0. 1 1 7 0. 20 1.0 P "nos acrw i (9027) 50 50 1. 0 2. 0 0. 2 17 20 5O Salmonella schottmuclleri (9149) 50 50 4.0 0.2 0.1 4. 0 1. 1 '3. 3 Proteus vulgaris (8427) 50 50 0.2 0.1 0.1 17 2.0 1.0 Salmonella typhosa (167) 50 50 1. 0 0.1 0.02 2 5 .67 1, 2

1 The antibiotic described and claimed in parent copending application Serial No. 238,072, filed July 23, 1951.

An aerosol spray containing amicetin A, or a salt thereof, as the essential ingredient is useful in hospitals, especially in tuberculosis wards, to prevent or reduce the possibility of transmission of pathogenic organisms such as M. tuberculosis (H37Rv) and the like from patient to patient or from patient to doctor. Likewise, wash solutions containing amicetin A or a salt thereof are also useful for general sanitation purposes, for example, washing of hands or cleaning of equipment, floors or furnishings in tuberculosis wards, and also in laboratories experimenting with mycobacteria.

@The toxicity of various antibiotics, including amicetin A, is given in the following table:

trum is determined in aqueous solution, .maxima of at 272 millimicrons .are observed. When the spectrum Eir...=s41 at millimicrons and E;z-,,=2ss

at 313.millimicrons are observed. The specific rotation oflthe anhydrous amicetin A in'0.1 Nhydrochloric acid at [a] =plus'138.8 degrees, at a concentration of 0.5 percent. r Figure, 2*shows the infrared absorption spectrum made from a suspension of anhydrous crystalline amicetin A mulled in liquid petrolatum. It shows the following characteristic absorption bands in the infrared; expressed iii-reciprocal centimeters: 3430, 3340,3225, 1650, 1567, 1604,1480, 1337, 1303, 1175, 1132, 1087,1055, 1040, 993, 938, 888, 843, 788, 764, 694. The individual bands at about 3430,3340, and'3225 are characteristic of the 'and'N- -H groups. The bands at 1650 and 1567 are "characteristic of the mono-substituted amide carbonyl groups'p Thebands 'at1604 and 1480 are characteristic of the unsaturated ring systems.

The Rf value of amieetimA (the ratio of movement of the liquid front to the movement pfthe antibiotic), is about 0.64 when n-butanol saturated with water is employed as the developing agent. When a mixture of ninety percent of n-butanoland ten percent of Waterv is. used as the developing agent, an Rf value of about 0.43 is observed. 7

Movement of antibiotic substances producedby the fermentation of S. vinaceusedrappus was obtained, on

paper chromatography, according to the method of Peterson and Reineke, I. Chem. Soc, 72, 3598 (1950), using n-butanol saturated with water. The properties indicated above clearly show that amicetin A is different from any other known and previously characterized antibiotic material.

In order that the activity 'of the culture filtrates, extractiomfractions, and the crude, purified, or crystalline preparations ofamicetin A'. could be easily and routinely evaluated, an agar plate assay procedure wasemployed using avium"%(7992), asa test organism and purestreptomycin sulfate as-the assay standard. a An M. avium unit of activity (or'more simple; a unit of-activity) as herein used is definedas an equivalent of one microgram ofi pure streptomycin. free 13356., Theassay procedure is based on that of Loo et al., I. Bact., 50, 701 (1945).

The agar medium consistsofitrypticase soy broth, thirty grains; (trypticase is. a pepton derived from casein by pancreatic digestion); agar, twenty grams; beef extract, 3.0:grams;-"Iween."80, 0. 1" milliliter; and sufiicient water to make a volume of one liter. appliedto FAT-inch filter. paper disks (Whatman) and the plates are incubated at 37 degrees centigrade for sixteen toeightee'n hours, The range of the assay curve is from 6.25 units per milliliter to 100 units per milliliter.

The following examples illustrate the formation, recovery, concentration, purification, crystallization and identification of amicetin A These examples are merely ustrative in nature and are: not to be construed .as

EXAMPLE 1.-FORMATION AND RECOVERY OF AMICETINA' T6 "a SW-milliliterErlenmeyerflask is added 100mm.

liters of a seed medium containing the following in- Difcogbacto-peptone I 5 Difco, beef extract Sodium, chloride Tap water to1 000cc."

NOTE-D i;f9, bacto peptoneis a productof high pep- The test solution is tone andamino acid content containing a negligible quantity'only of proteoses and complex nitrogenous constituents. Difco, beef extract is a specifically prepared beef extract standardized to give uniform results. Itis prepared by cooking waste meat products in water, filterinEQJand concentrating the filtrate to the consistency of a paste.

- The flask and its contents are sterilized by autoclaving I for a period of twenty minutes at a temperature of 120 degrees centigrade. After cooling, the flask is inoculated witha loopful of S. vinaceous-drappus spore suspension obtained from, a casein-starch agar slant and the inoculated flask is shaken on'a reciprocating shaker at the rate of ninety four-inch strokes ,perminute for a period of 24 hours at a' temperature of thirty degrees centigrade.

At the end of this period, 25 milliliters of the vegeta tive growth thus-obtained is used to inoculate a 22-liter sweepstirrer seed bottle charged withtwelve liters of they above-indicated medium. (Prior to inoculation, the cons tainer and itsflcontents are sterilized by autoclaving for 7 onehour at a temperature of 120degrees centigrade.)

The inoculated medium is fermented with an air flow of tenliters per, minute and at a temperature of 26 degrees centigrade.- At the end of a 48-hour period, the fer Tap water to l'liter.

Thetank is sterilized prior to inoculationby heating for aperiod of twenty minutes at a temperature of 120' The fermentation is conducted at 28 degrees Centigrade and the medium: aerated at a rate The fermentation.

degrees centigrade.

ofabout'220 cubic feet per hour. medium isharvested after a period of 88 hours.

A' 7.5-liter aliquot portion of the filtered harvested beer (assaying 83 M. avium. units per milliliter) is treated. witl12250 gramsof sodium chloride and extracted at a pH'of 9.3 with two 1500-mil1iliter portions of amyl acetate. (Spent beer assay shows that eighty percent of the" activityqis transferred to the amyl acetate portion.) Thejamyl' acetate solution containing the antibiotic activity is successively extracted with 200 milliliters of 0.15 N hydrochloricacid and two 200-milliliter portions of 01075 M hydrochloric acid.. The aqueous solutions.

thus obtained are combined, treated with 180 grams of sodium chloride, and extracted at a pH of 9.2 with three' seventy-milliliter portions of amyl acetate. The organic extracts are combined, clarified by filtration, and ex ,tracted with three 25 -milliliter portions of 0.1 N hydrochloric acid. The combined aqueous extract is adjusted toa pI-Iof 8,6,concentrated in vacuo to a volume of 4 about forty millliters and, after refrigerating for three days, 383 milligrams. of crude crystalline amicetin A is obtained. The product thus obtained is further purified by dissolving in methanol, decolorizing with activated charcoal, adding water thereto and allowing the solution to crystallize. The purified crystals. of amicetin A thus obtained are pale yellow in color, assay 425 M. aviztm unitsper milligramand melt between 209 and 212 de grees centigrade on a Kofler microhotstage. The following results are obtained on analysis:

Analysis.- -Calc. for C H N O C, 59.41; H, 6.76; N, 13.41. Found: C, 59.03, 59.24; H, 6.00, 6.74; N, 1332,1352.

AMICETIN A 1136 500- milliliter Erlenmeyer flask is added mini literlsofi the seed medium described in Example 1, and Wtthef its contents are sterilized by autoclaving ZEXAMPLE 2. EORMATION AND RECOVERY on for twenty minutes at 120 degrees centigrade, After cooling, the flask is inoculated with a loopful of a Streptomyces vinaceus-drappus spore suspension obtained from a casein-starch agar slant, and the inoculated flask isincubated at thirty degrees Centigrade on areciprocating shaker operating at the rate of ninety four-inch strokes per minute. H

After a period of 48 hours, 25 milliliters of the formed vegetative growth is used to inoculate a five-gallon sweepstirrer charged with twelve liters of the sterilized medium indicated supra. The fermentation is conducted at a temperature of 26 degrees centigrade for a period of 48 hours with an air flow of ten liters per minute. The contents of the sweepstirrer are used to inoculate a 100-gallon tank charged with 240 liters of the medium described in Example 1. (Prior to inoculation, the tank and medium are sterilized by heating at a temperature of 120 degrees centigrade for twenty minutes.) The fermentation is conducted at a temperature of 32 degrees centigrade with any air flow of 150 cubic feet per hour.

After a period of 88 hours, the fermentation medium is harvested and the beer is filtered using Dicalite 4200 (a diatomaceous silica sold by The Great Lakes Carbon Corporation, Chicago, Illinois) as the filter aid. Sixtyfive gallons of filtered beer assaying 118 M. avz'um units per milliliter are obtained. The beer is adjusted to a pH of 8.0 to 8.5 with a sodium hydroxide solution followed by extraction in a Podbielniak extractor withsixty liters of n-butanol. The butanol solution is extracted batchwise with three six-liter portions of dilute sulfuric acid, the solution thus-obtained adjustedto-a pH of 8.2 and then extracted with n-butanol. The resulting solution is extracted with dilute acid and the acid extract thus-obtained is adjusted to a pH of 7.0 and freeze-dried'to yield 24.6 grams of a white powder assaying 230M. avium units per milligram. The crude product thus-ob tained is dissolved in 400 milliliters of 0.1 N hydrochloric acid, the solution is filtered and the filtrate adjusted to a pH of 6.6. The brown amorphous precipitate thus-obtained is removed by filtration, the filtrate is adjusted to a" pH of 8.4, seeded and then refrigerated overnight. crystalline material separates and is collected and dried. The product is recrystallized from methanol and water to yield 3.08 grams of amicetin A in the form of pale' yellow platelets melting at 209 to 212 degrees centigrade and assaying about 380 M. avium units per milligram.

EXAMPLE 3.-PREPARATION OF THE RIDE OF AMICETIN A Crystalline amicetin A (1.88 grams) is dissolved'in 100 milliliters of 0.05 N. hydrochloric acid. The result: ing pale yellow solution is filtered and freeze-dried. The

dried powder thus-obtained is dissolved in a solution containing ten milliliters of methanol, eight milliliters of water andtwo milliliters of 0.05 N hydrochloric acid. During the portionwise addition of 100 milliliters of acetone to the resulting solution, while crystals are formed. After cooling overnight, the crystals are collected and recrystal lized from a water-methanol mixture. A-yield of 1.62 grams of anicetin A hydrochloride is obtained assaying 350 M. avium units per milligram. 1

EXAMPLE 4.PREPARATION OF THE HELIANTHATE OF AMICETIN A A solution of 56 milligrams of amicetin A in 25 milliliters of dilute hydrochloric acid 'is adjusted'to pH 5.6 by careful addition of a dilute sodium hydroxide solution. To this solution is added 101 milligrams'of methyl orange dissolved in 25 milliliters of warm water. A precipitate separates slowly. After three hours of cooling at four degrees centigrade, the crystalline precipitate is collected and dried to yield 98 milligrams of the helianthate ,of amicetin A. About 85 milligrams of this product is'recrystallized from aqueous methanol to yield 65 milli- HYDROCHLO-' grams of a red-orange crystalline material which exhibits decomposes above 210 degrees en'tigfad'e. The lielianthate of anicetin A thus-prepared assays 167 M. avia'm' units per milligram.

Analysis.Calc. for C H N Q S C, 57.26; H, 5;87; N, 13.58; S, 5.18. Found: C, 56.92, 56.49; H, 6.46, 6.89; N, 13.54; S, 4.30.

It is to be understood that the invention is not to" be limited to the exact details of operation or exact compounds shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art and the invention is therefore to be limited only by the scope of the appended claims. 1

We claim:

' 1. Amicetin A, a substance characterized by being efof forming salts with acids, and sparingly soluble in water and methanol, and in its essentially pure crystalline form by meltingbetween 209 and 212degrees centigrade by having an empirical formula corresponding ,to' the following analysis: C, 59.24; H, 6.74; N, 13.52; by having an ultraviolet absorption spectrum in aqueous solution exhibiting maxima of I I lZ".;..=' 0 at 251 millimicrons and at 320 r'nillimicronsand minima of at 230 millimicrons and Ettsi'ifi at 272 millimicrons" and, in a 0.1 N hydrochloric acid solution, maxima of I lzin. j

at 256 millimicrons and I I Eit...=2's's at 313 millimicrons; by exhibiting a characteristic absorption in the infrared region of the spectrum'when sus-' pended in mineral oil, at the followingfrequencies'expressed in reciprocal centimeters. 3430, 3340, 3225, 1650; 1604, 1567, 1480, 1337, 1303, 1175, 1132, 1087, 1055, 1040, 993, 938, 888; 843, 788', 764; 694; and by having a specific'rotation [al in 0.1 N'hydrochloric acid solution'of plus 138.8 degrees'at a concentration of 0.5 percent. I '2. A substance selected from the class consisting of amicetin A as characterized in-claim 1 and'the acid addition salts thereof.

3. An acid addition 'salt'of amicetin'Aas characterized i Waksman et al.: The Actinomycetes and Their Antibiotics, pub. Wilkins and Williams, Balt;,.Md., pp. 170 184', 190, 217.

Sawozski: J. of Antibiotics (Japan), vol. 8, No, 2, Ser.,

A, pp. 39-41.

Klarman: Am. J. Pharm., July 1955, pp; 252-254.

Stevens 6n: Nature," September '25, 1954, p. 598-599; 1

Johnson ct al.: waksmaa'etat: seineaMarch'zs; 1949Lv'1. 109 N5." 2830, pp. 305-307.

otherrerreneesafirbnowing age Sobin July is, 1950- 10 Hinman et a1.: J.A.C.S., vol. 75, 1953, pp. 499, 500, 5864-5871.

McCormick ct al.: Antibiotics and Chemotherapy, July 1953, pp. 718720. 

1. AMICETIN A, A SUBSTANCE CHARACTERIZED BY BEING EFFECTIVE IN INHIBITING THE GROWTH OF MYCOBACTERIA, CAPABLE OF FORMING SALTS WITH ACIDS, AND SPARINGLY SOLUBLE IN WATER AND METHANOL, AND IN ITS ESSENTIALLY PURE CRYSTALLINE FORM AND MELTING BETWEEN 209 AND 212 DEGREES CENTIGRADE BY HAVING AN EMPRICAL FORMULA CORRESPONDING TO THE FOLLOWING ANALYSIS: C, 59-24: H,6.74; N, 13.52: BY HAVING AN ULTRAVIOLET ABSORPTION SPECTRUM IN AQUEOUS SOLUTION EXHIBITING MAXIMA OF 